Small Size in Living Cells

Estimating microbial growth

Describe the various methods used to estimate the amount of microbial growth that has taken place in a culture:
a. viable/standard plate count
b. serial dilutions
c. turbidity measurement
d. direct microscopic count
e. coulter counter
f. membrane filtration

a. viable/standard plate count

The viable count is done by plating the samples onto appropriate culture medium and counting the number of cell or colonies formed from the plates. (1) First you have to dilute your sample to 10 fold and if you count that there is 20 colonies or bacteria cells from the colonies. You can calculate how many viable cell by multiplying the number of colonies 20 X 10 = 200 colonies. So the total number of viable cells is 200 colonies forming units. The units for the viable cell count is cfu, which stands for colony forming units.

b. serial dilutions

A serial dilutions is performed in order to determine the how many bacteria colonies that are formed from the original cultures. It is usually performed before the viable plate cell count method. For example, the original culture containing bacteria are diluted by a serial dilutions series by taking 10ul of the original culture and add to 90 ul water. This is a 10-fold dilution (Dilution1) because it is dilutied 10X times. From the 10fold (dilution 1), you can take another 10 ul from dilution1 and and 90 ul of water, so you have 10X10 fold dilution, which is 100X Fold dilution (dilution2) because you diluted it two times serially. From the dilution 2, you can make another dilution by taking 10 ul from dilution 2 and add 90 ul water; this will be 10X10X10 dilution (dilution 3). You can make further dilution from dilution 3 like, 10 ul (dilution 3) and 90 ul water to yield dilution 4 ( 10 X 10 X10 X10). If you stop here, then you will have a 1 X 10e4 dilution. If you plate the aliquot of this dilution 4, and you get 20 colonies or cells. You can determine the number of original bacterial cells in your culture by multiplying 20 X 10X10X10X10 = 200000 colonies in your bacteria cultures.

c. turbidity measurement

Turbidity method is a quick and easy method to measure the turbidity or "cloudiness" of a culture and " translate the cloudiness into cell numbers". (1) This method is prefered when a large number of cultures are counted. The turbidity of the culture must be correlated with the initial cell number. You can do this by determining the cloudiness of different concentrations of a given bacteria in the medium and use a standare plate count to determine how many viable colonies contribute to the amount of turbidity. If you have 20 colonies, then you measure how turbid that is by the absorbance(nm). For example, if 20 colonies measured a turbidity of 500 nm, the 10 colonies should give you smaller number such as 250 nm. 5 colonies should give you a turbidity of 150 nm. You will construct a standard curve correlating the amount of colonies on the x axis and the amount of turbidity by the y axis. From this standard curve, you can extrapolate how many cells you have by measuring the turbidity. The turbidity can be measured using an instrument called the calorimeter or sphectrophotometer to measure the light source that travel through the bacteria cells. (1)

d. direct microscopic count

A "Petroff-Hauser" counting chambers is used to count the number of bacteria cells in a culture or liquid medium. You inoculate an aliquot of your culture onto the counting chambers and using a microscope, you count the number of cells in each chamber. There are four chambers in the counting chamber. So you just count how many cells you have.
If you have 5 per chambers, than you would have 5 X4 = 20 cells or colonies. It is not possible to distinguish dead cells from live cells so the viable cell count is not reliable in this method. (1) The advantage is that it is a fast method. (1)

e. coulter counter

The coulter counter is a instrument that uses the " Electrical Sensing Zone" method for counting cells. It works by using two electrodes; one inside the aperature and one outside the aperture. The two electrodes create an electrical field and a disturbance between the two electric fields is measured when the cells or particles pass through the aperture. (2) When the bacteria particles pass through the aperture, they cause a change in the electric field as a "voltage pulse". (2) The concentration of particles can be counted this way by the measurement of the voltage pulse.

f. Membrane filtration

Membrane filtration is really a method to "separate particles from a liquid form for the purpose of purifying bacteria from the liquid solution. " (3) The membrane filter has a pores of a particular size that can separate bacteria of a particular size from the cultures. The membrane pores has sizes that is small enough to filter out larger particles than bacteria. So the pores of the membrane only allow the bacteria to filter through, but left particles larger than bacteria to stay behind and not filter through. The outside of the solutions contain larger particles than bacteria and the filtrate only contain small particles like bacteria .(3)

View Full Posting Details